The investigation of the major determinants of monoamine synthesis and turnover in vivo is of scientific interest because monoamine levels in the central nervous system (CNS) play critical roles in neuropsychiatric, neuroendocrine and cardiovascular diseases. Tyrosine and tryptohan hydroxylase are known to be the rate-limiting steps in the synthesis of catecholamines and serotonin, respectively. Current evidence suggests that the in vivo rate of synthesis of these compounds may be mediated by the concentration of the hydroxylase cofactor, tetrahydrobiopterin (BH4). We have previously reported a significant positive correlation between hydroxylase cofactor content and total hydroxylase enzyme activity across rat brain areas. In addition, there is an unusually high content of cofactor in certain neuroendocrine tissues, which may indicate a potential neuroendocrine role for the cofactor. Using the catecholaminergic neurotoxin, 6-hydroxydopamine, we now report greater than 85% reduction in reduced cofactor content in both the substantia nigra and striatum after intranigral injection. Kainic acid injected in the striatum, which destroys striatal cell bodies and interneurons, caused a modest decrease in striatal cofactor. These results indicate that reduced cofactor is highly concentrated within dopaminergic neurons of the nigrostriatal system.